Characterization of a broad pH range protease of Candida caseinolytica M. Poza, T. de Miguel, C. Sieiro 1 and T.G. Villa Department of Microbiology and Parasitology, Faculty of Pharmacy, University of Santiago de Compostela, Spain; 1 Present address: Department of Microbiology, Faculty of Sciences, University of Vigo, 36200 Vigo, Spain 815/5/01: received 25 April 2001 and accepted 16 May 2001 M. POZA, T. DE MIGUEL, C. SIEIRO AND T. G. VILLA. 2001. Aims: The study of a protease secreted by Candida caseinolytica for use in future industrial applications. Methods and Results: Growth of Candida caseinolytica on a medium containing milk induced a rapid production of an extracellular enzyme able to hydrolyse casein. The crude extract was applied to both Sephacryl S-200 and DEAE-Biogel A columns, obtaining one peak of activity showing a molecular mass of » 30 kDa and three active peaks, respectively. These four peaks showed the same biochemical parameters. In all cases, an extremely broad pH range of action was determined. Conclusions: Candida caseinolytica secretes high levels of an extracellular protease when grown either in rotary shakers or in batch-fermenters. Signi®cance and Impact of the Study: The biochemical properties of this enzyme suggest its possible industrial application in the brewing industry, in the formulation of certain type of detergents and in the fur and leather industries, among others. INTRODUCTION Proteases are the single class of enzymes that occupy a pivotal position with respect to their applications in both the physiological and commercial ®elds. Their vast diver- sity and speci®c range of action have attracted the attention of biotechnologists worldwide. Although they are widely distributed in nature, micro-organisms are the preferred source of these enzymes in fermentation bioprocesses because of their fast growth rate and also because they can be genetically engineered to generate new enzymes with desirable abilities or simply for enzyme overproduc- tion (North 1982; Mala Rao et al. 1998). Strong proteolytic activity is relatively rare in yeast. However, several authors have found some strong activities in different yeast species (Ogrydziak 1993). The screening of a large number of microorganisms with industrial applications in milk treat- ment was boosted due to the high nutritional value of milk and many studies have been carried out to search for activities of interest. Different caseinolytic yeasts have been found, including some species of Kluyveromyces and Candida, such as C. punicea, C. lipolytica, C. aquatica and C. curiosa (Ahearn et al. 1968). Candida caseinolytica was ®rst isolated during a screening of more than 5000 microorganisms from necrotic tissues of several Opuntia and Stenocereus cactus species growing in a restricted region of the North American Sonoran Desert and a few other localities in Baja California, Mexico. In fact, this yeast is the only cactus-speci®c species that exhibits strong extracellular caseinolytic activity and it also differs from other species of the genus in both morphological and physiological properties (Phaff et al. 1994). In the present work we report on the isolation and the basic biochemical properties of the enzymes involved in the degradation of casein produced by C. caseinolytica strain UCD-FST 83±438á3. MATERIALS AND METHODS Strains, media and culture conditions The yeast used in this work was Candida caseinolytica, type strain UCD-FST 83±438á3. Milk medium (MM) containing beef extract 0á3%, bactotryptone 0á5%, glucose 0á1%, agar Correspondence to: T.G. Villa, Department of Microbiology and Parasitology, Faculty of Pharmacy, University of Santiago de Compostela, 15706 Santiago de Compostela, Spain (e-mail: mpvilla@usc.es). ã 2001 The Society for Applied Microbiology Journal of Applied Microbiology 2001, 91, 916±921